miRCURY LNA miRNA Power Target Site Blockers

For studying the effects of an individual miRNA on a single target site

Custom-designed target site blockers for specific inhibition of miRNA targets

Sophisticated design
and superior high affinity, regardless of target sequence

Unmatched high efficacy in vitro and in vivo

Unrivaled performance and high protein expression due to lack of RNase H-dependent mRNA degradation

Efficient at very low concentrations, outcompeting miRNAs for their target sites

Superior biological stability for long-lasting antisense activity

miRCURY LNA miRNA Power Target Site Blockers are antisense oligonucleotides that bind to the miRNA target site of an mRNA, preventing miRNAs from gaining access to that site. This enables you to study the effects of an miRNA on a single target. miRCURY LNA miRNA Power Target Site Blockers are efficient at very low concentrations, and due to their high affinity from LNA enhancement, the target site blockers outcompete miRNAs for their target sites.

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15 nmol miRCURY LNA miRNA Power Target Site Blockers, in vivo Ready, with option of different labels and purifications, provided in tube format

miRCURY LNA miRNA Power Target Site Blockers are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

miRNA inhibitors vs. target site blockers.

The phenotype observed when inhibiting an miRNA can be due to many targets being upregulated. Target site blockers (TSB) lead to the upregulation of one specific target and may or may not cause the same phenotype.

Examples of target site blocker applications for pathways X and Y.

Examples of target site blocker applications for pathway Z.

The CAV1 target site blocker phenocopies the miR-199a-5p inhibitor in many important aspects.

miR-199a-5p stimulates TGFbeta signaling by turning down CAV1 expression. Inhibiting miR-199a-5p directly using a miRNA inhibitor (LNA-miR-199a-5p) or inhibiting the interaction of miR-199a-5p with CAV1 using a target site blocker (CAV1 protector), reduces TGFbeta signaling and causes several phenotypes. (C) Both inhibition of miR-199a-5p or using the CAV1 target site blocker (TSB) reduces myofibroblast differentiation as visualized by alpha-smooth muscle actin (a-SMA) staining (green). (D) Co-transfection of the miR-199a-5p inhibitor as well as the CAV1 TSB reduces expression from a SMAD-luciferase reporter plasmid. (E and F) Both inhibition of miR-199a-5p or using the CAV1 TSB reduces the migration rate of TGFbeta stimulated hFL1 lung fibroblasts leading to slower wound closure. From: Cardenas CL, et al. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1. PLoS Genet. 2013;9(2):e1003291.

(A) Target site blockers are antisense oligonucleotides designed to compete with miRNA/RISC by hybridizing to the miRNA target site of a particular mRNA. (B) LNA-enhanced, high-affinity target site blockers compete effectively with miRNA/RISC for the miRNA target site. In addition, LNA distribution throughout the LNA/DNA mixmer ensures that the antisense oligonucleotide does not catalyze RNase H-dependent degradation of the mRNA. As a result, the TSB will cause increased expression of the protein encoded by the targeted mRNA.

Performance

Unravel miRNA function with target site blockers

miRNAs typically regulate gene expression of multiple targets, and inhibition of an miRNA will result in derepression of all of these targets. Therefore, a phenotype observed upon miRNA inhibition is a composite result of derepression of several targets (see figure miRNA Inhibitors vs. target site blockers). However, deregulation of a few of these targets will often contribute significantly to the phenotype. Identifying these targets is important to understanding the function of the miRNA.

miRCURY LNA miRNA Power Target Site Blockers can be used to:

Determine which pathway is involved in a phenotype observed upon miRNA inhibition

Determine which miRNA/mRNA interactions are most important in a pathway containing several predicted targets

What are target site blockers?

miRCURY LNA miRNA Target Site Blockers are LNA-enhanced antisense oligonucleotides that bind to the miRNA target site of an mRNA, thereby preventing miRNAs from gaining access to that site. This allows researchers to study the effects of an miRNA on a single target. In contrast, the phenotype observed when inhibiting an miRNA reflects the combined effects of that miRNA on all targets.

LNA-enhanced target site blockers

The incorporation of LNA into the miRCURY LNA miRNA Power Target Site Blocker means that they will compete more effectively with the miRNA/RISC complex for the miRNA target site. In addition, LNA distribution throughout the LNA/DNA mixmer ensures that the antisense oligonucleotide does not catalyze RNase H-dependent degradation of the mRNA. As a result, the TSB will cause increased expression of the protein encoded by the targeted mRNA by preventing miRNA-mediated translational attenuation (see figure LNA-enhanced target site blockers compete effectively with RISC for miRNA binding site).

Procedure

Following resuspension, miCURY LNA miRNA Target Site Blockers are transfected into cells with a transfection reagent or via electroporation. Phenotypic effects of the target site blocker are normally assessed 24–72 hours after transfection. For some applications, such as cell differentiation assays, the phenotypic readout may take place 7–10 days after transfection.

Applications

miRCURY LNA miRNA Target Site Blockers can be used for:

Determining which pathway is involved in a phenotype observed upon miRNA inhibition

Determining which miRNA/mRNA interactions are most important in a pathway containing several predicted targets